With the rapid development of and increasing need for monoclonal and polyclonal antibodies for both diagnostic and therapeutic purposes, research efforts have recently been directed to devising new techniques for effectively isolating antibodies from antibody-containing solutions. Some well-known, existing techniques include the use of classic liquid chromatography adsorbents, such as ion exchangers, hydrophobic supports, hydroxy apatite and gel filtration media. Unfortunately, these techniques are time-consuming and tedious to perform and cannot be applied generally to the isolation of heterogenous populations of antibodies. As a result, such techniques need to be adjusted on a case-by-case basis depending upon the specific antibody sought to be isolated.
Affinity chromatography, which relies on specific interactions between an immobilized ligand and a particular molecule sought to be purified, is another well-known, existing technique used to purify antibodies from solution. Protein A, which is derived from the bacterium Staphylococcus aureus, has a strong and specific affinity for the Fc fragment of IgG antibodies and has been used for approximately one decade as an affinity ligand for purifying IgG antibodies. Protein A can be immobilized on a large variety of solid support materials, such as chromatographic beads and membranes, and can be used to obtain high purity antibodies in high yield.
Unfortunately, Protein A suffers from a number of limitations as an affinity ligand. One such limitation is the prohibitive cost of Protein A. Because of its high cost, the large scale utilization of Protein A as an affinity ligand is not often economically feasible. Another such limitation is the sensitivity of Protein A to those proteases that are commonly present in the same biological fluids (e.g., sera, ascitic fluids, milk, hybridoma cell culture supernatants, and the like) in which the antibodies being sought to be purified by affinity chromatography are also present. This sensitivity of Protein A to such proteases often leads to one or both of the following effects: (1) The recognition of Protein A towards the Fc fragments of IgG is progressively reduced; and (2) Fragments of Protein A generated by protease action contaminate otherwise pure antibody preparations.
Still another limitation with Protein A is that it is possible for Protein A to be released intact from its associated solid support material, thereby contaminating antibody preparations brought into contact therewith. The contamination of therapeutic antibody preparations with traces of Protein A or Protein A fragments is very serious not only because Protein A and/or Protein A fragments are capable of provoking an antigenic response in humans but also because Protein A is known as a potent mitogen.
Protein A is not the only protein which has been used as an affinity ligand for the purification of a class of antibodies. Protein G has similarly been used as an affinity ligand. See Bjorck et al., J. Immunol., 133:969 (1984). Protein G also interacts with the Fc fragment of immunoglobulins and is particularly effective in isolating mouse IgG antibodies of class 1 (as contrasted with Protein A which is not very effective in isolating these antibodies). Protein G, however, suffers from the same types of limitations discussed above in connection with Protein A.
Recently, a third protein has been identified as an effective affinity ligand for purifying antibodies. This protein is Protein L from Peptostreptococcus magnus. Protein L, as contrasted with Protein A and Protein G, interacts specifically with the light chains of IgG antibodies without interfering with their antigert binding sites. This specificity permits Protein L to complex not only with antibodies of the IgG class but also with antibodies of the IgA and IgM classes. Despite its broad affinity, Protein L suffers from the same limitations described above in connection with Proteins A and G.
In addition to suffering from the aforementioned limitations, Proteins A, G and L are all sensitive to a number of chemical and physical agents (e.g., extreme pH, detergents, chaotropics, high temperature) which are frequently used to clean affinity chromatography columns between runs. Consequently, some people have chosen to minimize the number of cleaning cycles applied to the above-described columns so as to correspondingly minimize degradation thereto. One drawback to this tactic, however, is that failure to clean the columns regularly prevents optimal antibody purification.
Anti-antibodies represent still another type of affinity ligand used for gamma globulin purification. Anti-antibodies, however, are limited in use due to their high cost and very limited stability.
In addition to the above-described protein-based affinity ligands, there are numerous lower molecular weight pseudobioaffinity (i.e., less specific) ligands which have been used for antibody purification. Histidine, pyridine and related compounds represent one type of pseudobioaffinity ligand commonly used for antibody purification. See e.g., Hu et al., "Histidine-ligand chromatography of proteins: Multiple modes of binding mechanism," Journal of Chromatography, 646:31-35 (1993); EI-Kak et al., "Interaction of immunoglobulin G with immobilized histidine: mechanistic and kinetic aspects," Journal of Chromatography, 604:29-37 (1992); Wu et al., "Separation of immunoglobulin G by high-performance pseudo-bioaffinity chromatography with immobilized histidine," Journal of Chromatography, 584:35-41 (1992); EI-Kak et al., "Study of the separation of mouse monoclonal antibodies by pseudobioaffinity chromatography using matrix-linked histidine and histamine," Journal of Chromatography, 570:29-41 (1991), all of which are incorporated herein by reference. See generally U.S. Pat. Nos. 5,185,313, 5,141,966, 4,701,500 and 4,381,239, all of which are incorporated herein by reference. However, non-specific binding of proteins and low capacity are the major limitations to adsorbents employing the above-identified compounds.
Thiophilic compounds represent another class of pseudobioaffinity ligands. An adsorbent utilizing one type of thiophilic compound is disclosed by Porath et al. in FEBS Lett., 185:306 (1985), which is incorporated herein by reference. This type of adsorbent is produced by reacting either a hydroxyl- or thiol-containing support first with divinyl sulfone and then with mercaptoethanol. The aforementioned adsorbent utilizes a salt-promoted promoted approach to adsorb immunoglobulins. Elution of adsorbed immunoglobulins is effected by decreasing salt concentration and/or by modifying pH.
Another type of pseudobioaffinity adsorbent capable of adsorbing antibodies utilizes mercaptopyridine as its ligand. See Oscarsson et al., "Protein Chromatography with Pyridine- and Alkyl-Thioether-Based Agarose Adsorbents," Journal of Chromatography, 499:235-247 (1990), which is incorporated herein by reference. This type of adsorbent is generated, for example, by reacting mercaptopyridine with a properly activated solid support. The adsorbent thus formed is capable of adsorbing antibodies under high salt conditions.
Other pseudobioaffinity adsorbents utilizing thiophilic compounds are described in the following patents and publications, all of which are incorporated herein by reference: U.S. Pat. No. 4,897,467; published PCT Application No. PCT/U.S. Pat. No. 89/02329; published European Patent Application No. 168,363; Oscarsson et al., "Thiophilic adsorbents for RIA and ELISA procedures," Journal of Immunological Methods, 143:143-149 (1991); and Porath et al., "A New King of `Thiophilic` Electron-Donor-Acceptor Adsorbent," Makromol. Chem., Macromol. Symp., 17:359-371 (1988).
The above-described thiophilic adsorbents provide a generally satisfactory means for purifying antibodies; however, in those cases in which the initial biological liquid is a protein rich solution, such as a serum or ascites, the non-specific binding by such adsorbents of a number of proteins other than the desired antibodies can be a problem. This problem of non-specific binding is the primary limitation of these thiophilic adsorbents.
Another group of low molecular weight ligands capable of selectively binding antibodies includes pentafluoropyridine and N-dimethylaminopyridine reacted with ethylene glycol, glycine or mercaptoethanol. See Ngo, J. Chromatogr., 510:281 (1990), which is incorporated herein by reference. Adsorbents utilizing these materials can be used to isolate immunoglobulins in either high salt or low salt buffers or to isolate other types of proteins under low salt conditions. Elution of adsorbed proteins can be obtained by lowering pH.
Still other low molecular weight pseudobioaffinity ligands have been identified as being capable of selectively binding antibodies from egg yolk and other biological liquids. These ligands are special dyes. Elution of the bound antibodies from the ligands is achieved by special displacers.
All of the above-mentioned adsorbents utilizing low molecular weight pseudobioaffinity ligands are very attractive in terms of their low cost and their chemical and physical stability. However, their level of non-specific binding and/or their toxicity (should they, for example, contaminate a therapeutic antibody preparation intended for administration to humans) are too high, and their capacity for antibodies is too low to counterbalance the attractiveness of adsorbents utilizing Proteins A, G or L as specific antibody ligands.